Fatty acid and phospholipid synthesis is a vital facet of bacterial physiology that is tightly regulated at several levels. Phospholipids comprise approximately 15% of the dry weight of a bacterial cell and the advantage of maintaining fine control over their biosynthesis is evident from the importance of phospholipid composition in membrane structure and function. Our experimental plan proposes to investigate the regulation of bacterial lipid metabolism at three key points. The first control point is at the initiation of fatty acid biosynthesis. The evidence indicates that an early step in the fatty acid biosynthetic pathway is rate-limiting. The initiation of fatty acid synthesis is more complicated, potentially utilizing three distinct mechanisms. The two key enzymes are beta-ketoacyl-ACP synthase III and acetyl-CoA:ACP transacylase. We have cloned and characterized the synthase III (fabH) gene providing the necessary tools to critically test the role of this enzyme in initiation. Virtually nothing is known about the genetics or biochemistry of acetyl transacylase and a major effort to acquire the molecular reagents to establish the role of acetyl transacylase in fatty acid initiation is the next important step. Control mechanisms that regulate fatty acid composition are located at later points in the fatty acid biosynthetic pathway. Unsaturated fatty acids (UFA) are critical components that are essential for cell survival and modulate the activity of membrane enzymes and transport systems. The sophisticated regulation of UFA synthesis has been extensively investigated and is known to involve two genes of fatty acid biosynthesis (fabA and fabB) and the transcriptional regulator, fadR. We have identified and cloned a fourth gene, designated fabJ, that is required for UFA biosynthesis. Characterizing the structure and function of the fabJ gene product is essential to understanding the mechanisms that govern UFA production. A third level of control is exerted by 2-acyl-GPE acyltransferase/acyl- ACP synthetase, an acyltransferase that maintains membrane phospholipid structure by recycling lysophospholipids that are generated as a byproduct in the synthesis of other molecules or by the action of phospholipases. This enzyme is also thought to be responsible for the uptake and incorporation of exogenous fatty acids and lysophospholipids into the membrane. We have isolated mutants and cloned the structural gene (aas) for this enzyme thus providing the molecular tools needed to define the structure and function of this membrane-associated acyltransferase.